One of the defense mechanisms against infection by both animals and plants is the production of peptides that have antimicrobial and antiviral activity. Various classes of these peptides have been isolated from tissues both of plants and animals. One well known class of such peptides is the tachyplesins which were first isolated from the hemocytes of the horseshoe crab as described by Nakamura, T. et al. J Biol Chem (1988) 263:16709-16713. This article described the initial tachyplesin isolated from the Japanese species, Tachyplesin I, which is a 17-amino acid amidated peptide containing four cysteine residues providing two intramolecular cystine bonds. In a later article by this group, Miyata, T. et al. J Biochem (1989) 106:663-668, extends the studies to the American horseshoe crab and isolated a second tachyplesin, Tachyplesin II, consisting of 17 residues amidated at the C-terminus, also containing four cysteine residues and two intramolecular disulfide bonds. Two additional 18-mers, called polyphemusins, highly homologous to Tachyplesin II and containing the same positions for the four cysteine residues, were also isolated. Polyphemusin I and Polyphemusin II differ from each other only in the replacement of one arginine residue by a lysine. All of the peptides were described as having antifungal and antibacterial activity. A later article by Murakami, T. et al. Chemotherapy (1991) 37:327-334, describes the antiviral activity of the tachyplesins with respect to vesicular stomatus virus; Herpes Simplex Virus I & II, Adenovirus I, Reovirus II and Poliovirus I were resistant to inactivation by Tachyplesin I. Morimoto, M. et al. Chemotherapy (1991) 37:206-211, found that Tachyplesin I was inhibitory to Human Immunodeficiency Virus. This anti-HIV activity was found also to be possessed by a synthetic analog of Polyphemusin II as described by Nakashima, H. et al. Antimicrobial Agents and Chemotherapy (1992) 1249-1255. Antiviral peptides have also been found in rabbit leukocytes as reported by Lehrer, R. I. et al. J Virol (1985) 54:467-472.
Another class of antimicrobial and antiviral peptides, the "protegrins" have been isolated from porcine leukocytes as reported by the present applicants in a paper by Kokryakov, V. N. et al. FEBS (1993) 337:231-236. An additional paper disclosing cationic peptides from porcine neutrophils was published by Mirgorodskaya, O. A. et al. FEBS (1993) 330:339-342. Storici, P. et al. Biochem Biophys Res Comm (1993) 196:1363-1367, report the recovery of a DNA sequence which encodes a pig leukocyte antimicrobial peptide with a cathelin-like prosequence and is reported to be one of the protegrins.
Other important classes of cysteine-containing antimicrobial peptides include the defensins, .beta.-defensins and insect defensins. The defensins are somewhat longer peptides characterized by six invariant cysteines and three intramolecular cystine disulfide bonds. Defensins were described by Lehrer, R. I. et al. Cell (1991) 64:229-230; Lehrer, R. I. et al. Ann Rev Immunol (1993) 11:105-128. A review of mammalian-derived defensins by Lehrer, R. I. et al. is found in Annual Review Immunol (1993) 11:105-128; three patents have issued on the defensins: U.S. Pat. Nos. 4,705,777; 4,659,692; and U.S. Pat. No. 4,543,252. Defensins have been found in the polymorphonucleated neutrophils (PMN) of humans and of several other animals, as well as in rabbit pulmonary alveolar macrophages, and in murine small intestinal epithelial (Paneth) cells and in corresponding cells in humans.
.beta.-Defensins are found in bovine neutrophils and respiratory epithelial cells. See Selsted, M. E. et al. J Biol Chem (1993) 288:6641-6648, Tang, Y-Q. J Biol Chem (1993) 268:6649-6653, and Diamond, G. et al. Proc Natl Acad Sci (USA) (1991) 88:3952-3958, Diamond, G. et al. Proc Natl Acad Sci USA (1993) 90:4596-4600. Insect defensins have been reported by Lambert, J. et al. Proc Natl Acad Sci (USA) (1989) 88:262-265.
Antifungal and antibacterial peptides and proteins have also been found in plants (Broekaert, W. F. et al. Biochemistry (1992) 31:4308-4314) as reviewed by Cornelissen, B. J. C. et al. Plant Physiol (1993) 101:709-712. Expression systems for the production of such peptides have been used to transform plants to protect the plants against such infection as described, for example, by Haln, R. et al. Nature (1993) 361:153-156.
The present invention relates to an additional group of antimicrobial peptides with broad spectrum activity, representative members of which can be isolated from chicken neutrophils. These peptides, named gallinacins, contain six cysteine residues in a pattern reminiscent of the .beta.-defensins and the tracheal antibiotic peptide (TAP) derived from bovine respiratory epithelial cells. These peptides contain 35-40 amino acids and are useful as preservatives and therapeutics.